The germ line maintains the genetic continuity of the species while the soma is responsible for the growth and development of the individual organism. Physical differences between the genomes of the germ line and the soma are essential for their respective roles. The somatic macronucleus of the ciliated protozoan, Tetrahymena, develops from a mitotic product of the germ line micronucleus during sexual reproduction. Macronuclear development involves DNA methylation, DNA rearrangement and transcriptional activation of the macronuclear genome. We will investigate the biological role and mechanism of inheritance of DNA methylation in the macronuclear DNA of Tetrahymena. Transcriptional regulation: Previous studies on the pattern of methylation near structural genes will be extended to additional genes. Ribosomal RNA genes will be hypermethylated in vitro and microinjected into vegetative cells. DNA rearrangement: The relative timing of DNA methylation and rearrangement in the developing macronucleus will be ascertained. Inheritance of methylation: We will determine whether nucleosome phasing occurs in regions of the genome which are uniformly methylated and whether a site which is methylated in vivo is methylated when it is present at a different position in the genome. We will ask whether methylation patterns introduced in vitro are maintained when the DNA is microinjected into host cells and whether there are hemimethylated sites in newly replicated DNA of dividing cells.